Citrus Tristeza Virus Amit Levy [email protected] 863-956-8704 Brief History:

• CTV existed in Asia for centuries, with growers adapting tolerant varieties • Out of Asia, citrus got heavily infected with Phytophthora sp., which was managed by adapting the more tolerant sour orange rootstock. • The result- citrus production in many areas was almost entirely based that single rootstock. • This decision had grave effects when CTV pandemics swept throughout the world, causing ‘quick decline’ (death) of trees on this rootstock. • Since the 1930's that the extent of this deadly disease problem manifested itself first in Argentina and shortly later in Brazil and California, with the death of millions of trees • Switching to non-sour orange rootstock eliminated the danger from CTV decline. • But now, with HLB, growers are switching back… Citrus Tristeza Virus (CTV) – Decline

CTV decline is associated with death of phloem near the bud union, resulting in a girdling effect that may cause the overgrowth of the scion at the bud union, loss of feeder roots and thus drought sensitivity, stunting, yellowing of leaves, reduced fruit size, poor growth, dieback, wilting, and death. Roots of declining tree – no feeder roots Citrus tristeza virus (CTV) – decline

•Transmission and Epidemiology aphid transmitted by a series of aphids, brown citrus aphid best also melon, cotton aphids transmitted in a semi-persistent manner – ~ 1 hr to ~ 24 hr

•Identification in field, identify rootstock – sour orange bud union staining, scion overgrowth, thickened bark at union serology – ELISA detect CTV; can differentiate FL mild from decline PCRs biological assay in greenhouse vein clearing and leaf cupping in Mexican lime

•Control use alternative rootstock use virus-free or decline-free budwood Citrus Tristeza Virus (CTV) – Stem Pitting

Stem pitting results in pits in the wood under depressed areas of bark and are often associated with severe stunting and considerably reduced fruit production.

FS674 on DG

Duncan Graprfruit Citrus tristeza virus (CTV) – stem pitting

no severe stem pitting isolates of CTV known to be in Florida (moderate isolates are here) every effort is being made to keep them out

•Host Range and Symptoms stem pitting does not kill trees reduces vigor, reduces growth, reduces yield and fruit size stem pitting is specific to virus isolate and host some isolates cause stem pitting in grapefruit some isolates cause stem pitting in sweet orange some isolates cause stem pitting in both In Florida, mandarins are more tolerant

•Control keep stem pitting isolates out mild strain cross protection The Causal agent: citrus tristeza virus (CTV) CTV Virion: • long flexuous particles, 2000nm long × 10–12 nm in width. • The virions have helical symmetry with about 8-9 capsids per helix turn, and a central hole of 3-4 nm. Gold-labeled CP antiserum

Gold-labeled CPm antiserum

CTV virions From: Tian et al. J. Gen. Virol.80:1111 (1999)

p25 (major capsid protein, CP) p27 (minor capsid protein, CPm) CTV Genome: • positive-stranded RNA virus ~ 20,000 nts- CTV • CTV genome is divided almost equally into two parts, • the 5' part consisting of ORF1a and 1b harboring the viral replication machinery • the 3' end half harboring 10 ORFs encoding a range of structural components and other gene products involved in virion assembly and host and vector interactions.

structural components + other products Replication- not very conserved involved in virion assembly and host and vector interactions- very conserved

RdRp p6 p61 p18 p20

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p33 HSP70 p27 CP p13 p23

ORF1a+1b ORF 2-11 12 Open Reading Frames (ORFs) are expressed through a variety of mechanisms

ORF 1a 1b 2 3 4 5 6 7 8 9 10 11

PRO PRO p33 HSP70h CPm p18 p20 MT IDR HEL ’ 5 probe 3’ probe RdRp p6 p61 CP p13 p23

( ) p33 1. Polyprotein: Replicase ( ) p6 ( ) HSP70h ( ) p61 ( ) CPm 2. +1 Ribosomal frameshift: RdRp ( ) CP ( ) p18 ( ) p13 ( ) p20 ( ) p23

3. Sub Genomic RNAs

Proteins • ORF 1a and 1b required (and sufficient) for replication • p33 (ORF 2) is required for superinfection exclusion and is required for infection of a subset of the viral host range • 6 kDa hydrophobic protein (ORF3) is a single-span transmembrane protein not required for virus replication or assembly, but it is required for infection of all plants of the host range. • 65 kDa cellular heat-shock protein homolog (HSP70h, ORF4), 61 kDa protein (ORF5), and the tandem pair of p27 (CPm, ORF6) followed by p25 (CP, ORF7) required for efficient virion assembly • CP is also a suppressor of RNA silencing. • The function of p18 (ORF 8) and p13 (ORF 9) are required for infection of certain hosts • Protein p20 (ORF 10) accumulates in amorphous inclusion bodies of CTV-infected cells and has been shown to be a suppressor of RNA silencing. • p23 (ORF 11) is a multifunctional protein with no homologue in other Closteroviruses , that: (i) binds cooperatively both single-stranded and dsRNA molecules in a non-sequence-specific manner (ii) contains a zinc finger domain that regulates the synthesis of the plus- and minus-strand molecules and controls the accumulation of plus-strand RNA during replication (iii) is an inducer of CTV-like symptoms in transgenic C. aurantifolia plants and (iv) is a potent suppressor of intracellular RNA silencing and (v) controls the level of negative-stranded of genomic an subgenomic RNAs. CTV Movement CTV is a phloem limited virus- only systemic movement

CTV-GFP Aniline blue CTV moves from sieve elements into phloem associated cells, where it replicates

GFP Antibody Phloem associated Phloem cells associated cells

Sieve element CTV moves from sieve elements into phloem associated cells, where it replicates CP Antibody Phloem CP Antibody associated cells Phloem associated cells

Sieve element Replication, assembly

Particle Move CTV Control: • Spraying pepticide to control vector (Aphids) • Avoiding sour orange rootstocks

• Cross Protection:

• Superinfection exclusion is a phenomenon in which an established virus infection prevents the subsequent infection by another virus. • With CTV, only isolates from the same strain exclude other members of that strain. • If the challenging virus is of a different strain, it multiplies and spreads throughout the plant with no interference from the preexisting virus. • If the challenging virus is of the same strain as the established virus, it is excluded and does not become a component of the population. • Cross-protection is a practice in which a mild population of CTV is purposely pre- inoculated into trees to prevent infection and disease caused by endemic isolates. • Superinfection exclusion is dependent on the p33 gene and/or the 5’ protease.

CTV as a tool: A B C Figure 2. A- Cartoon representation of a psyllid acquiring RNAi by feeding. The actual effect of CTV induced VIGS on the establishment of psyllids on control plants (B) versus CTV expressing a truncated JHMAT (ACP gene) /PDS fusion sequence (C)